The mu opioid receptor, the type most closely associated with both analgesia and behavioral reinforcement, is subject to a variety of factors that regulate its effectiveness in eliciting a cellular response. Among these are the structure of the receptor itself, its density in the cell membrane, and its proximity to coupling proteins. In addition, these factors are all linked by dynamic interactions whose timing, only partially understood at present, can have important influence on the performance of a drug. During the past year we have investigated these factors and dynamics in three ways. The first employed confocal microscopy and a high affinity fluorescent antagonist, "6-BNX", to investigate the location and dynamics of the receptors in their membranes. The second is the study of the irreversible antagonist beta-FNA to determine whether a mutant receptor, H297Q, remains capable of covalent modification by the drug and how its response to the drug differs from that of the wild-type receptor. The third is the establishment of the "macropatch" technique for recording the kinetics of drug transduction at a sub-second time scale at a few square micrometers of plasma membrane.The results of the three programs follow: (1) The studies with 6-BNX revealed that nonspecific binding was variable and could be high and misleading. However under defined conditions that take advantage of the slow dissociation rate of the probe from receptors, we could eliminate the nonspecific binding. Kinetic studies showed that 6-BNX specifically labeled receptors with a lag phase that indicated sequestration of the probe in a non-visible compartment, not yet identified, preceded the labeling of the receptors. (2) The studies with beta-FNA showed that this compound covalently bound to both wild-type and mutant H297Q receptors, confirming that the mutation has not radically reorganized the recognition pocket. As do other alkaloid antagonists, beta-FNA acted as a partial agonist at the mutant receptors. Its EC-50 was 1.8 nM, and its intrinsic activity was 0.35. The binding kinetics of radiolabeled beta-FNA illuminated an important clue, namely that the forward rate constant for the reversible association step was diminished by about 20-fold at the mutant receptor while the other two rate constants (dissociation and covalent binding) were unchanged, indicating the presence of a kinetic barrier to association in the mutant receptor. (3) The lab's main electrophysiology rig was reconfigured for macropatch recording from Xenopus oocytes. Cell attached, inside-out, and outside-out recordings of membrane patches have been successful, and single channel potassium currents, which are activated by the mu receptor, possessing appropriate conductance were observed. However, outside-out responses to opioids have not yet been obtained.